1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
Foreign animal disesaes, such as Foot-and-Mouth Disease Virus (FMDV), Classical Swine Fever (CSF), African Swine Fever (ASF), rinderpest (RP, Contagious Bovine Pleuropneumonia (CBPP) and lumpy skin disease (LSD) may enter the United States through various means, including deliberate introduction. Foreign animal diseaes (FAD) are a likely choice for terrorists in that many of the disease agents are easily aquirable, dissemination is not difficult, they are readily transmissable, and the livestock population is highly susceptible, and the economic impact of a major outbreak would be catastrophic.

To counter the new threat concers, the U.S. Department of Agriculture is developing a system of rapid real-time PCR assays for selected OIE class A animal pathogens to enable rapid detection and identification of diseased animals and effective managment of disease outbreaks resulting from accidential or intentional introduction into the United States.

2.List by year the currently approved milestones (indicators of research progress)
FY 03 Develop a standardized experimental design for the field validation of veterinary diagnostic assays.

FY 04 Estimate diagnostic specificity of the FMD RT-PCR for Texas beef cattle, swine, and sheep.

FY 05 Estimate diagnostic specificity of the CSF RT-PCR for Texas swine.

Estimate diagnostic sensitivity and specificity for FMD in endemic countries.

FY 06 Estimate diagnostic sensitivity and specificity for diagnosis of FMD virus infection in an acute outbreak situation (should one occur).

Develop methods to detect and differentiate Mycoplasma spp. of cattle usingReal-Time PCR and/or host gene expression biosignatures.

4b.List other significant research accomplishment(s), if any.
Archived samples from negative bovine & porcine cohorts for future reference in test evaluations.

Expert, transfer and implementation of RT-PCR equipment, RT-PCR test kits for FMDV, produced by TetraCore, and assay technology to an international laboratory (Brasil).

4c.List significant activities that support special target populations.
Nothing to report.

4d.Progress report.
The state of Texas was divided into 11 ecologic zones for estimation of the specificity of the FMDV RT-PCR in the negative cohort of cattle. The number of cattle necessary from each region is proportional to the cattle density as estimated by the National Agricultural Statistics Service, July 2003, and the samples were collected as follows: • Northern high plains (TAES Amarillo); 260 cattle • Southern high plains (Lubbock); 39 cattle • Low plains (TAES Vernon); 50 cattle • Low plains (Vernon); 39 cattle • Cross timbers (TAES Stephenville); 112 cattle • Blacklands (TAES Riesel); 124 cattle • North East Texas (TAES Overton); 115 cattle • South East Texas and Upper coast (Diboll); 52 cattle • Trans-Pecos (TAES El Paso); 27 cattle • Plateau (TAES Uvalde); 131 cattle • South Central and Coastal Bend (TAES Beeville); 141 cattle • Total cattle collected = 1090 cattle

The disease-free swine cohort for estimation of the specificities of the FMDV and CSFV RT-PCRs were collected from a slaughter plant in Texas. • 1100 samples completed 7/30/04

Extraction and FMD RT-PCR testing of 1,090 negative cohort cattle resulted in 32 positive or inconclusive samples being detected. Eleven of which were blinded-positive controls, and the remaining 21 questionable samples were sent to PIADC for further evaluation. All FMD test results from the negative bovine cohort have been compiled and submitted to USDA.

Samples were collected from 701 cattle representing unexposed, FMD-vaccinated-unexposed, FMD-vaccinated-FMDV-exposed, FMDV-exposed, and FMDV-infected populations from Amazonas, Mato Grosso do Sul, and Minas Gerais in Brasil. All samples were extracted and RT-PCR tested for FMDV, 16 of which were RT-PCR positive and some of which were confirmed positive by viral isolation and ELISA antigen capture. Lack of availability of dry ice to preserve the samples under very demanding and adverse field conditions may have compromised the quality of the results from 469 samples stored at 4°C for 1-7 days, yet positive RT-PCR, viral isolation and ELISA antigen capture results were obtained from two of these samples. Use of the FMD RT-PCR in Brasil was found to be a valuable diagnostic assay, but its application in remote areas was difficult due to limited availability of dry ice, therefore, alternatives (such as RNA Later™) for RNA conservation at room or even environmental temperatures would significantly improve the field applicability. Impact: This research is being utilized in the validation of real time PCR tests by APHIS.

This research falls within the component 1: Biodefense Research of the NP-103 National Program.

5.Describe the major accomplishments to date and their predicted or actual impact.
Completion and submission of all FMDV RT-PCR results to USDA from 1,100 swine and 1,108 cattle from negative cohort populations in Texas.

Completion and submission of all CSF RT-PCR results to USDA from 1,100 swine from negative cohort populations in Texas.

Completion of international transfer of technology for evaluation of the assay in 701 cattle from unexposed and FMDV-exposed populations from three states of Brasil.

6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Data from all FMD and CSF RT-PCR tests have been transferred to USDA scientists for evaluation of assay specificity, sensitivity, precision and accuracy with the ultimate goal being test approval and use as an official test in approved laboratories.

RT-PCR test kits for CSFV, produced by TetraCore, Inc. have been approved by USDA, APHIS.

7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
None.